Figure 1.
TcdB induces necrosis in epithelial cells.
A, TcdB does not induce caspase-3/7 activation in HeLa cells, as detected by a fluorescent indicator, Apo-One. B, TcdB induces rapid death in HeLa cells, as detected by a luminescent indicator, CellTiterGlo. Caspase-3/7 activation and viability values represent the average of three experiments in which each condition was tested in triplicate. The error bars indicate the standard deviation between three experiments. C, HeLa cells were synchronized and incubated with or without TcdB for 2.5 hours at 37°C. A representative image obtained by light microscopy indicates rounding in cells treated with 10 pM TcdB and a loss of membrane integrity in cells treated with 10 nM TcdB. D, Extracellular LDH was detected in TcdB-treated HeLa cells after 2.5 hours using a luminescence-based indicator, Cytotox-Glo. Increased levels of LDH release were apparent after 8 hours. LDH release values represent the average of three experiments in which three replicates were averaged. Error bars indicate the standard deviation between the values obtained from the three experiments. E, HeLa cells were treated with a buffer control or 10 nM TcdB for 1 h and then fixed with 4% formaldehyde. Cells were stained with an antibody specific for HMGB1 and an Alexa Fluor 488 anti-mouse antibody. The cells were visualized with a LSM510 Confocal microscope. The representative images show that HMGBI is released from the nucleus of HeLa cells when treated with 10 nM TcdB and remains nuclear in the untreated cells.
Figure 2.
Mutations in the autoprocessing domain active site and the cleavage site result in TcdB proteins with impaired autoprocessing activity in vitro and in cells.
A, Autoprocessing was induced in vitro by incubating wild-type TcdB and TcdB mutants with multiple InsP6 concentrations and 1 mM DTT at 37°C. After 2 hours, the proteins were subjected to SDS-PAGE and visualized with Coomassie stain. A representative series of gels is shown from experiments performed in triplicate. B, Three replicates of the experiments shown in panel A were quantified by densitometry. Bands corresponding to TcdB 544–2366 were quantified and normalized to the band corresponding to TcdB 1–2366 without InsP6. Error bars reflect the standard deviation of the percent cleavage between three experiments. The data indicate that wild-type TcdB autoproteolysis can be detected at concentrations of 1 uM to 1 mM InsP6. By comparison, TcdB mutants C698S, C698A, and H653A were completely inactive for autoprocessing at all InsP6 concentrations. The TcdB D587N and L543A had some residual activity, but autoprocessing activity was impaired relative to wild-type. C, GTDs of autoprocessing mutants are not released in cells. HeLa cells were synchronized for 30 minutes at 4°C, then intoxicated with 10 nM toxin. Intoxicated cells were incubated at 4°C for an hour before being moved to 37°C. Cells were harvested after 50 minutes, and cell lysates were prepared for SDS PAGE and Western blot. The blot was probed with antibodies against the TcdB GTD, unglucosylated Rac1, total Rac1, and GAPDH. While release of the GTD in cells intoxicated with wild-type TcdB was detected, the free GTD was not detected in cells treated with autoprocessing deficient mutants. The absence of signal with an antibody that recognizes unglucosylated Rac1 suggests that the autoprocessing mutants are still able to modify Rac1 in cells.
Figure 3.
Autoprocessing mutants induce necrosis in epithelial cells.
A, Toxins were applied to HeLa cells at concentrations ranging from 0.1 pM to 100 nM, and viability was measured after 2.5 hours with CellTiterGlo. B, Toxins were applied to Caco2 cells at concentrations ranging from 0.1 pM to 100 nM, and viability was measured after 18 hours with CellTiterGlo. Percent viability was determined by normalizing the signal from treated cells to the signal from untreated cells. C, Autoprocessing mutants did not induce caspase-3/7 activation after 25 hours. D, Comparable levels of extracellular LDH were detected after 8 hours in HeLa cells treated with wild-type TcdB and TcdB autoprocessing mutants. In each panel, the values represent the average of three experiments in which three replicates were averaged. Error bars indicate the standard deviation between the values obtained from the three experiments.
Figure 4.
TcdB glucosyltransferase mutants cause epithelial cell death.
A, TcdB and TcdB glucosyltransferase domain mutants (100 nM) were tested for their capacity to glucosylate purified Rac1 (2 uM) in the presence of 20 mM UDP-[14C]-glucose over the course of 1 h. The proteins were resolved by SDS-PAGE, and the gels were analyzed by phosphorimaging. B, TcdB D270N was tested with higher concentrations of both toxin and UDP-[14C]-glucose. Only at the highest concentrations of both toxin and UDP-[14C]-glucose is residual activity apparent. C, The TcdB glucosyltransferase mutants are impaired in their glucosyltransferase activities in HeLa cells, as determined by Western and an antibody specific for unglucosylated Rac1. D, Wild-type TcdB and the TcdB glucosyltransferase mutants induced comparable levels of HeLa cell death, as determined by CellTiterGlo, after 2.5 h of treatment. Percent viability was determined by normalizing the signal from treated cells to the signal from untreated cells. Values reflect the average signal from three experiments in which each condition was tested in triplicate. Error bars correspond to the standard deviation in the percent viability from the three experiments.
Figure 5.
TcdB and TcdB autoprocessing mutants cause cell rounding with concentration dependent kinetics.
HeLa cells were treated with multiple concentrations of wild-type and mutant TcdB proteins and imaged every 10 minutes over a 2 hour time course. The percentage of round cells was quantified over six fields for each concentration and time point. The kinetics of rounding induced by TcdB and the TcdB autoprocessing mutants is shown at concentrations of A, 10 pM. B, 100 fM and C, 1 fM. D, Representative images of cells treated with 1 fM TcdB and TcdB autoprocessing mutants for 50 minutes. Green cells are alive; red cells are dead. Images were collected with an Opera High-Throughput Confocal Screening Microscope in an environment-controlled chamber at 37°C, 5% CO2. Round cells were defined as having an area less than 500 um2 and a width-to-length ratio of less than 0.4. Analysis was performed using Columbus Analysis software.
Figure 6.
TcdB and TcdB C698A cause epithelial damage in porcine colonic explants.
Porcine colonic explants were treated with 1 mM DTT to remove the mucus layer, washed with PBS, and incubated with toxin at 37°C for 5 hours. A, Tissue sections were stained with H&E. B, The H&E slides were scored in a blinded fashion using a semi-quantitative injury scale: 0- no damage; 1-superficial damage, damage limited to intact surface epithelial cells; 2-loss of up to 50% of surface epithelial cells or gland length, crypts intact; 3-loss of over 50% of surface epithelial cells and damage in greater than 50% of gland length. An injury score was calculated as the mean score for sections analyzed seven times by six individuals. Statistical analysis was performed using a two-way ANOVA and post-hoc tests. These analyses revealed a significant difference in the scores given to tissues treated with the toxins over the range of concentrations (p<0.001), while there was no statistical difference between tissues treated with TcdB and TcdB C698A. A subsequent Bonferroni's test revealed that scores given to tissue treated with 10 nM TcdB and 10 nM TcdB C698A were significantly different from scores given to tissue treated with 10 pM TcdB or 10 pM TcdB C698A (p<0.001). Error bars correspond to the standard deviation between the seven scores. C, The sections were also stained with an anti-pan keratin and D, anti-active caspase-3 antibody. Representative images of H&E, pan-cytokeratin, and active caspase-3 staining (white – pan-cytokeratin/active caspase-3, green – DAPI) show significant damage to the epithelium of the colon at concentrations of TcdB and TcdB C698A that kill cells (10 nM). At concentrations that induce rounding but not death in cultured cells (10 pM), there was no significant damage to the tissue surface cells. Caspase-3 activation was not detected at levels above background in any of the TcdB-treated tissues.